Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations.

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

Integrative miRNA-mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy.

Personalized management of neuropathic pain is an unmet clinical need due to heterogeneity of the underlying aetiologies, incompletely understood pathophysiological mechanisms and limited efficacy of existing treatments. Recent studies on microRNA in pain preclinical models have begun to yield insights into pain-related mechanisms, identifying nociception-related species differences and pinpointing potential drug candidates. With the aim of bridging the translational gap towards the clinic, we generated a human pain-related integrative miRNA and mRNA molecular profile of the epidermis, the tissue hosting small nerve fibres, in a deeply phenotyped cohort of patients with sodium channel-related painful neuropathy not responding to currently available therapies. We identified four miRNAs strongly discriminating patients from healthy individuals, confirming their effect on differentially expressed gene targets driving peripheral sensory transduction, transmission, modulation and post-transcriptional modifications, with strong effects on gene targets including NEDD4. We identified a complex epidermal miRNA-mRNA network based on tissue-specific experimental data suggesting a cross-talk between epidermal cells and axons in neuropathy pain. Using immunofluorescence assay and confocal microscopy, we observed that Nav1.7 signal intensity in keratinocytes strongly inversely correlated with NEDD4 expression that was downregulated by miR-30 family, suggesting post-transcriptional fine tuning of pain-related protein expression. Our targeted molecular profiling advances the understanding of specific neuropathic pain fine signatures and may accelerate process towards personalized medicine in patients with neuropathic pain.

Recent advances for using human induced-pluripotent stem cells as pain-in-a-dish models of neuropathic pain.

Neuropathic pain is amongst the most common non-communicable disorders and the poor effectiveness of current treatment is an unmet need. Although pain is a universal experience, there are significant inter-individual phenotypic differences. Developing models that can accurately recapitulate the clinical pain features is crucial to better understand underlying pathophysiological mechanisms and find innovative treatments. Current data from heterologous expression systems that investigate properties of specific molecules involved in pain signaling, and from animal models, show limited success with their translation into the development of novel treatments for pain. This is in part because they do not recapitulate the native environment in which a particular molecule functions, and due to species-specific differences in the properties of several key molecules that are involved in pain signaling. The limited availability of post-mortem tissue, in particular dorsal root ganglia (DRG), has hampered research using human cells in pre-clinical studies. Human induced-pluripotent stem cells (iPSCs) have emerged as an exciting alternative platform to study patient-specific diseases. Sensory neurons that are derived from iPSCs (iPSC-SNs) have provided new avenues towards elucidating peripheral pathophysiological mechanisms, the potential for development of personalized treatments, and as a cell-based system for high-throughput screening for discovering novel analgesics. Nevertheless, reprogramming and differentiation protocols to obtain nociceptors have mostly yielded immature homogenous cell populations that do not recapitulate the heterogeneity of native sensory neurons. To close the gap between native human tissue and iPSCs, alternative strategies have been developed. We will review here recent developments in differentiating iPSC-SNs and their use in pre-clinical translational studies. Direct conversion of stem cells into the cells of interest has provided a more cost- and time-saving method to improve reproducibility and diversity of sensory cell types. Furthermore, multi-cellular strategies that mimic in vivo microenvironments for cell maturation, by improving cell contact and communication (co-cultures), reproducing the organ complexity and architecture (three-dimensional organoid), and providing iPSCs with the full spatiotemporal context and nutrients needed for acquiring a mature phenotype (xenotransplantation), have led to functional sensory neuron-like systems. Finally, this review touches on novel prospective strategies, including fluorescent-tracking to select the differentiated neurons of relevance, and dynamic clamp, an electrophysiological method that allows direct manipulation of ionic conductances that are missing in iPSC-SNs.

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

ABSTRACT: Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.